The application of efficient non-viral gene transfer systems is desirable in modern DNA-vaccine design and gene therapy protocols. Therefore, many administration modes and formulations are under investigation to ensure optimal delivery of plasmid DNA for gene transfer. A significant improvement in the application of plasmid based DNA molecules in this field is the use of so-called minicircle-DNA.
Conventional plasmid vectors include a bacterial backbone and a transcription unit. The transcription unit carries the target gene or sequence (e.g. a sequence coding for a therapeutically useful protein) along with necessary regulatory elements needed for a specific gene transfer application. [1] The bacterial backbone unit includes inter alia elements which are needed for the stable propagation of plasmid-DNA in bacterial cells. [1] The latter elements, i.e. an antibiotic resistance gene as well as a bacterial replication origins but also unmethylated CpG motives or cryptic expression signals are clearly undesired for clinical applications of DNA-molecules in humans.
To remove these unwanted elements without destroying the supercoiled structure of the transcription unit, the minicircle technology has been developed [2-5]. This technology creates a minimal expression cassette by an in vivo site-specific recombination process which removes all unwanted backbone elements. In the course of this process, a so-called parental plasmid is divided by a recombinase into a miniplasmid carrying the backbone sequences and a minicircle consisting of almost exclusively the desired expression cassette (minimal expression cassette).
Following the site-specific recombination process the resulting mixture of plasmid species (i.e. minicircles, miniplasmides and to some extend unrecombined parental plasmid) must be separated, to isolate the desired minicircle-DNA. Different strategies have been developed for this purpose, including affinity based chromatographic purification and in vivo restriction. A successful approach to affinity based chromatographic purification has been described by Mayrhofer et al. [6]. Another method for minicircle purification, i.e. the in vivo restriction approach has been described by Chen et al. and Kay et al. [7, 8].
Chen and coworkers developed a technique to degrade the miniplasmid and remaining (unrecombined) parental plasmid in vivo via co-expression of a restriction enzyme [7]. The homing endonuclease used in this approach (I-SceI) and the PhiC31 integrase (i.e. the recombinase) are both located on the parental plasmid. Both genes are under the control of a PBAD/araC arabinose promoter. Thus, addition of the inducer L-arabinose results in the simultaneous expression of both the integrase and the endonuclease. However, when using this approach, even after 240 minutes of co-expression of integrase and endonuclease, contaminating miniplasmids and parental plasmids still make up about 3% of the total plasmid DNA [7]. In addition to that, the simultaneous expression of both the endonuclease and the integrase leads to undesired early degradation of the parental plasmid, thus reducing the minicircle yield obtained by said process. Various measures, including change of pH and temperature of the culture broth during the production process, were necessary to minimize premature degradation of the parental plasmid [7].
Another in vivo restriction approach described by Kay et al. [8] uses a bacterial producer strain expressing both the PhiC31 integrase (i.e. the recombinase) and a I-SceI homing endonuclease, whereby multiple copies of both enzymes which are under the control of the inducible promoter system PBAD/araC too, have been integrated into the chromosome of said producer strain. Although for this system fewer contaminations of about 1.5% are reported (compared to up to 15% with the system described by Chen et al. [7] as stated in [8]), it also requires change of both pH-value and temperature in the culture broth during production, thus making a large scale production process difficult. Furthermore, as bacteria tend to lose redundant, unnecessary genomic information, stability problems of the producer strain may occur.
Hence, there remains a need for methods for the production of minicircles achieving high yields of minicircles, allowing for an efficient isolation of the minicircles produced and being suitable for large scale production.